Many disorders result from inappropriate inflammation, including atherosclerosis, ischemia-reperfusion injury after heart attack, and asthma, among others. Recent studies have shown two routes used by leukocytes to enter tissues via the endothelium: between endothelial cells via the paracellular route, and directly through endothelial cells via the transcellular route. In the transcellular route a transmigratory cup (TC) forms on the surface of the endothelial cell, surrounding and engulfing leukocytes as they travel through endothelial cells. The signaling events during transcellular transendothelial migration (TEM), particularly in the formation of TCs, remain largely unknown. The major goal of this proposal is to understand the spatiotemporal coordination of Rho family GTPases and their regulation by Rho Guanine Exchange Factors (GEFs) in the formation of TCs during leukocyte TEM in inflammation. Our preliminary data and the known role of Rho GTPases in controlling cytoskeletal behavior leads me to hypothesize that RhoG, Rac1, and RhoA must be activated with precise timing and localization to form TCs, each playing a different role in cytoskeletal rearrangement, and Rho GEFs that strongly activate RhoG and/or Rac1, Vav2, Vav3, Trio, and SGEF, mediate this coordination. Thus, the specific aims of this proposal are: 1) to determine the spatiotemporal dynamics of RhoG, Rac1, and RhoA activity in TC formation during leukocyte TEM and 2) to determine the role of RhoGselective Rho GEFs in TC formation during leukocyte TEM. Using antibody-mediated clustering of ICAM-1 in COS-7 cells, human umbilical vein endothelial cells, and human microvascular endothelial cells, I will examine a Rho GTPase signaling network mediating TC formation. I will design and use unique tools that permit visualization of RhoG, Rac1, and RhoA conformational changes in individual living cells. These tools, together with mutation and siRNA manipulation of GTPases and GEFs, will elucidate the precise control of the cytoskeleton for TC formation in TEM. This information may help in the development of new therapies for inflammatory diseases such as atherosclerosis and ischemia-reperfusion injury. Lay Summary: In a variety of inflammatory diseases, including cardiovascular disease among others, the immune system's white blood cells enter tissues inappropriately and cause damage, leading to subsequent disability or death. The experiments proposed in this grant will help understand how blood cells leave the bloodstream and enter the tissues, which could lead to new therapies for preventing white blood cells from entering the vasculature or heart inappropriately and thus better treatment of cardiovascular disease.